Pathways to Specialized Ribosomes: The Brussels Lecture

Abstract“Specialized ribosomes” is a topic of intense debate and research whose provenance can be traced to the earliest days of molecular biology. Here, the history of this idea is reviewed, and critical literature in which the specialized ribosomes have come to be presently defined is discussed. An argument supporting the evolution of a variety of ribosomes with specialized functions as a consequence of selective pressures acting on a near-infinite set of possible ribosomes is presented, leading to a discussion of how this may also serve as a biological buffering mechanism. The possible relationship between specialized ribosomes and human health is explored. A set of criteria and possible approaches are also presented to help guide the definitive identification of “specialized” ribosomes, and this is followed by a discussion of how synthetic biology approaches might be used to create new types of special ribosomes

rRNA mutants in the yeast peptidyltransferase center reveal allosteric information networks and mechanisms of drug resistance

To ensure accurate and rapid protein synthesis, nearby and distantly located functional regions of the ribosome must dynamically communicate and coordinate with one another through a series of information exchange networks. The ribosome is ∼2/3 rRNA and information should pass mostly through this medium. Here, two viable mutants located in the peptidyltransferase center (PTC) of yeast ribosomes were created using a yeast genetic system that enables stable production of ribosomes containing only mutant rRNAs. The specific mutants were C2820U (Escherichia coli C2452) and Ψ2922C (E. coli U2554). Biochemical and genetic analyses of these mutants suggest that they may trap the PTC in the ‘open’ or aa-tRNA bound conformation, decreasing peptidyl-tRNA binding. We suggest that these structural changes are manifested at the biological level by affecting large ribosomal subunit biogenesis, ribosomal subunit joining during initiation, susceptibility/resistance to peptidyltransferase inhibitors, and the ability of ribosomes to properly decode termination codons. These studies also add to our understanding of how information is transmitted both locally and over long distances through allosteric networks of rRNA–rRNA and rRNA–protein interactions

Torsional restraint: a new twist on frameshifting pseudoknots

mRNA pseudoknots have a stimulatory function in programmed −1 ribosomal frameshifting (−1 PRF). Though we previously presented a model for how mRNA pseudoknots might activate the mechanism for −1 PRF, it did not address the question of the role that they may play in positioning the mRNA relative to the ribosome in this process [E. P. Plant, K. L. M. Jacobs, J. W. Harger, A. Meskauskas, J. L. Jacobs, J. L. Baxter, A. N. Petrov and J. D. Dinman (2003) RNA, 9, 168–174]. A separate ‘torsional restraint’ model suggests that mRNA pseudoknots act to increase the fraction of ribosomes directed to pause with the upstream heptameric slippery site positioned at the ribosome's A- and P-decoding sites [J. D. Dinman (1995) Yeast, 11, 1115–1127]. Here, experiments using a series of ‘pseudo-pseudoknots’ having different degrees of rotational freedom were used to test this model. The results of this study support the mechanistic hypothesis that −1 ribosomal frameshifting is enhanced by torsional resistance of the mRNA pseudoknot

An Extensive Network of Information Flow through the B1b/c Intersubunit Bridge of the Yeast Ribosome

Yeast ribosomal proteins L11 and S18 form a dynamic intersubunit interaction called the B1b/c bridge. Recent high resolution images of the ribosome have enabled targeting of specific residues in this bridge to address how distantly separated regions within the large and small subunits of the ribosome communicate with each other. Mutations were generated in the L11 side of the B1b/c bridge with a particular focus on disrupting the opposing charge motifs that have previously been proposed to be involved in subunit ratcheting. Mutants had wide-ranging effects on cellular viability and translational fidelity, with the most pronounced phenotypes corresponding to amino acid changes resulting in alterations of local charge properties. Chemical protection studies of selected mutants revealed rRNA structural changes in both the large and small subunits. In the large subunit rRNA, structural changes mapped to Helices 39, 80, 82, 83, 84, and the peptidyltransferase center. In the small subunit rRNA, structural changes were identified in helices 30 and 42, located between S18 and the decoding center. The rRNA structural changes correlated with charge-specific alterations to the L11 side of the B1b/c bridge. These analyses underscore the importance of the opposing charge mechanism in mediating B1b/c bridge interactions and suggest an extensive network of information exchange between distinct regions of the large and small subunits

PRFdb: A database of computationally predicted eukaryotic programmed -1 ribosomal frameshift signals

The Programmed Ribosomal Frameshift Database (PRFdb) provides an interface to help researchers identify potential programmed -1 ribosomal frameshift (-1 PRF) signals in eukaryotic genes or sequences of interest. To identify putative -1 PRF signals, sequences are first imported from whole genomes or datasets, e.g. the yeast genome project and mammalian gene collection. They are then filtered through multiple algorithms to identify potential -1 PRF signals as defined by a heptameric slippery site followed by an mRNA pseudoknot. The significance of each candidate -1 PRF signal is evaluated by comparing the predicted thermodynamic stability (ΔG°) of the native mRNA sequence against a distribution of ΔG° values of a pool of randomized sequences derived from the original. The data have been compiled in a user-friendly, easily searchable relational database. The PRFdB enables members of the research community to determine whether genes that they are investigating contain potential -1 PRF signals, and can be used as a metasource of information for cross referencing with other databases. It is available on the web at http://dinmanlab.umd.edu/prfdb .https://doi.org/10.1186/1471-2164-9-33

Altering SARS Coronavirus Frameshift Efficiency Affects Genomic and Subgenomic RNA Production

In previous studies, differences in the amount of genomic and subgenomic RNA produced by coronaviruses with mutations in the programmed ribosomal frameshift signal of ORF1a/b were observed. It was not clear if these differences were due to changes in genomic sequence, the protein sequence or the frequency of frameshifting. Here, viruses with synonymous codon changes are shown to produce different ratios of genomic and subgenomic RNA. These findings demonstrate that the protein sequence is not the primary cause of altered genomic and subgenomic RNA production. The synonymous codon changes affect both the structure of the frameshift signal and frameshifting efficiency. Small differences in frameshifting efficiency result in dramatic differences in genomic RNA production and TCID50 suggesting that the frameshifting frequency must stay above a certain threshold for optimal virus production. The data suggest that either the RNA sequence or the ratio of viral proteins resulting from different levels of frameshifting affects viral replication

The many paths to frameshifting: kinetic modelling and analysis of the effects of different elongation steps on programmed –1 ribosomal frameshifting

Several important viruses including the human immunodeficiency virus type 1 (HIV-1) and the SARS-associated Coronavirus (SARS-CoV) employ programmed −1 ribosomal frameshifting (PRF) for their protein expression. Here, a kinetic framework is developed to describe −1 PRF. The model reveals three kinetic pathways to −1 PRF that yield two possible frameshift products: those incorporating zero frame encoded A-site tRNAs in the recoding site, and products incorporating −1 frame encoded A-site tRNAs. Using known kinetic rate constants, the individual contributions of different steps of the translation elongation cycle to −1 PRF and the ratio between two types of frameshift products were evaluated. A dual fluorescence reporter was employed in Escherichia coli to empirically test the model. Additionally, the study applied a novel mass spectrometry approach to quantify the ratios of the two frameshift products. A more detailed understanding of the mechanisms underlying −1 PRF may provide insight into developing antiviral therapeutics

Endogenous ribosomal frameshift signals operate as mRNA destabilizing elements through at least two molecular pathways in yeast

Although first discovered in viruses, previous studies have identified operational −1 ribosomal frameshifting (−1 RF) signals in eukaryotic genomic sequences, and suggested a role in mRNA stability. Here, four yeast −1 RF signals are shown to promote significant mRNA destabilization through the nonsense mediated mRNA decay pathway (NMD), and genetic evidence is presented suggesting that they may also operate through the no-go decay pathway (NGD) as well. Yeast EST2 mRNA is highly unstable and contains up to five −1 RF signals. Ablation of the −1 RF signals or of NMD stabilizes this mRNA, and changes in −1 RF efficiency have opposing effects on the steady-state abundance of the EST2 mRNA. These results demonstrate that endogenous −1 RF signals function as mRNA destabilizing elements through at least two molecular pathways in yeast. Consistent with current evolutionary theory, phylogenetic analyses suggest that −1 RF signals are rapidly evolving cis-acting regulatory elements. Identification of high confidence −1 RF signals in ∼10% of genes in all eukaryotic genomes surveyed suggests that −1 RF is a broadly used post-transcriptional regulator of gene expression

A molecular clamp ensures allosteric coordination of peptidyltransfer and ligand binding to the ribosomal A-site

Although the ribosome is mainly comprised of rRNA and many of its critical functions occur through RNA–RNA interactions, distinct domains of ribosomal proteins also participate in switching the ribosome between different conformational/functional states. Prior studies demonstrated that two extended domains of ribosomal protein L3 form an allosteric switch between the pre- and post-translocational states. Missing was an explanation for how the movements of these domains are communicated among the ribosome's functional centers. Here, a third domain of L3 called the basic thumb, that protrudes roughly perpendicular from the W-finger and is nestled in the center of a cagelike structure formed by elements from three separate domains of the large subunit rRNA is investigated. Mutagenesis of basically charged amino acids of the basic thumb to alanines followed by detailed analyses suggests that it acts as a molecular clamp, playing a role in allosterically communicating the ribosome's tRNA occupancy status to the elongation factor binding region and the peptidyltransferase center, facilitating coordination of their functions through the elongation cycle. The observation that these mutations affected translational fidelity, virus propagation and cell growth demonstrates how small structural changes at the atomic scale can propagate outward to broadly impact the biology of cell

A flexible loop in yeast ribosomal protein L11 coordinates P-site tRNA binding

High-resolution structures reveal that yeast ribosomal protein L11 and its bacterial/archael homologs called L5 contain a highly conserved, basically charged internal loop that interacts with the peptidyl-transfer RNA (tRNA) T-loop. We call this the L11 ‘P-site loop’. Chemical protection of wild-type ribosome shows that that the P-site loop is inherently flexible, i.e. it is extended into the ribosomal P-site when this is unoccupied by tRNA, while it is retracted into the terminal loop of 25S rRNA Helix 84 when the P-site is occupied. To further analyze the function of this structure, a series of mutants within the P-site loop were created and analyzed. A mutant that favors interaction of the P-site loop with the terminal loop of Helix 84 promoted increased affinity for peptidyl-tRNA, while another that favors its extension into the ribosomal P-site had the opposite effect. The two mutants also had opposing effects on binding of aa-tRNA to the ribosomal A-site, and downstream functional effects were observed on translational fidelity, drug resistance/hypersensitivity, virus maintenance and overall cell growth. These analyses suggest that the L11 P-site loop normally helps to optimize ribosome function by monitoring the occupancy status of the ribosomal P-site